Development of a synthetic promoter pipeline for safe and efficient gene therapy

Gene therapy has been successful across many diseases ranging from muscular dystrophies to retinopathies. However, many studies still report genotoxicity or off-target effects due to limited specificity. Our project aims to tackle this challenge by developing a pipeline for the creation of highly specific, efficient, and short promoter sequences providing the desired control over the supplied genes. For the promoter pipeline, genes exclusively expressed in the target cell type are screened for regulatory elements, which are then sliced and shuffled to form potential promoter sequences. After validating this pipeline in preliminary experiments using B cells, we now want to further develop and refine it for novel targets. For this purpose, we chose oligodendrocytes and GABAergic neurons, which have both been implicated in neurological diseases making them attractive targets for gene therapy. After creating a library of novel regulatory sequences, the potential promoters will be screened over multiple rounds and combinations yielding the highest and most specific gene expression in target cells will be selected. These promoters will then be optimized through machine learning-based algorithms. Lastly, their specificity and efficiency will be confirmed in human cerebral organoids using adeno-associated viruses. These promoters will be used to ensure safety and efficiency of gene therapies. Moreover, the refined pipeline will enable us to create highly specific promoters for many other target cells in a labor- and cost-effective manner.